Fig 1.
Role of RyfA in osmotic and oxidative stress resistance.
(A) Growth under conditions of osmotic stress. Strains were grown shaking in LB medium until mid-log phase (O.D.600, 0.6) and plated on LB agar and LB agar with 0.6 M NaCl and 0.6 M urea (see Methods). (B) Growth inhibition zones (mm) of UPEC CFT073, ryfA mutant and complemented strains to oxidative stress generating compounds. Strains were grown on either LB agar or (C) M9- glucose agar. ROI-generating compounds tested were 30% hydrogen peroxide (H2O2), 53 mM plumbagin, or 40 mM paraquat. (D) Resistance to hydrogen peroxide in LB broth. WT CFT073 and ryfA mutant strains were examined in LB medium containing 5mM H2O2 or an equivalent volume of Milli-Q water (H2O). Samples were collected every 5 minutes, diluted, and plated on LB agar to determine CFUs. The CFT073 ΔoxyR strain was used as a sensitive control strain. The results represent the means of replicate experiments for a minimum of three samples. Vertical bars represent the standard errors of the means. Statistical significance was calculated by one-way ANOVA (A to D): (*, P < 0.05; **, P < 0.005; ***, P < 0.0001).
Fig 2.
CFT073 produces RyfA in mid-logarithmic phase.
(A) Northern blot analysis using 10 mg of total RNA isolated from WT CFT073 following growth in LB medium at 37°C. Lack of signal in ΔryfA strain confirms absence of RyfA in the ΔryfA strain. (B) Quantitative real-time PCR (qRT-PCR) analysis demonstrating that RyfA is produced by wild-type CFT073 under the conditions tested. (C) Northern blot showing RyfA expression in a strain overexpressing RyfA.
Fig 3.
Genes significantly affected by deletion of ryfA.
RNA-seq analysis was performed on RNA samples (in triplicate) from strain CFT073 and CFT073 ΔryfA grown in LB at mid-log phase of growth (O.D. 0.6). (A) Heatmap of the entire data set (n = 6) of CFT073 and ΔryfA mutant. Each row of the heatmap represents the log2 fold values transformed with Z-score of 121 differentially expressed genes (upregulation, red, downregulation, yellow). Hierarchical grouping of differentially expressed genes shows clustering. Z-scores are computed on a gene-by-gene basis by subtracting the mean and then dividing by the standard deviation. Specific genes and changes are presented in Fig 3B. (B) Genes classified in different categories based on log2 fold-change difference. Genes upregulated (yellow rectangles) and downregulated (blue rectangles) by at least 1.7-fold were considered significant with P < 0.05. Dark blue is greater or equal to (≥) a 3-fold decrease. Light blue is a decrease from 3-fold to 1.7-fold. Light yellow is an increase from 1.7-fold to 3-fold. Dark yellow is a greater or equal to (≥) 3-fold increase. Genes are classified in different categories with colored squares: General stress responses in red, metabolism and sugar transport in black, metabolism and iron transport in grey, patho-specific in pink, flagella and motility in purple, fatty acids and respiratory chain in green, amino acid metabolism in white, curli and toxin-antitoxin system in orange, RNA in yellow and unknown function in azure. Specific values fold-changes are presented in S1 Table (see supplementary data).
Fig 4.
Effect of deletion of ryfA on type 1 fimbriae (pili) production and motility.
(A) Yeast agglutination titer demonstrating level of production of type 1 fimbriae in strains cultured to the mid-log phase and after overnight static growth in LB broth and in human urine. The Δfim strain was used as a negative control and showed no agglutination. (B) Western blot of fimbrial extracts of strains cultured to the mid-log phase of growth in LB broth. The CFT073 fim-locked ON strain was used as a positive control. (C) Expression of fimA gene by qRT-PCR in the ΔryfA strain compared to the WT CFT073 strain in LB, human urine and infected bladders. (D) Motility of CFT073, ryfA mutant and complemented strain on 0.25% soft or urine agar. Each box and scatter dot plot (min to max) represents the mean diameter of the motility zone. Results are the mean values and standard deviations for at least three biological experiments. Statistical significance was calculated by the Student t test (A, C, D): *, P < 0.05; **, P < 0.005; ***, P < 0.0001. NS, not significant.
Fig 5.
Role of ryfA in the murine model of ascending UTI and in P fimbriae (pili) production.
(A) Adherence of strain CFT073 and its derivatives to human 5637 bladder epithelial cells in the presence or absence of 2.5% α-d-mannopyranose was determined. (B) Electron microscopy of CFT073 and the ΔryfA mutant. Arrows show fimbriae on cell surfaces. (C) Production of Pap fimbriae by hemagglutination assay. Results are the mean values and standard deviations for four biological experiments. (D) Single-strain infections to compare wild-type strain CFT073 to ΔryfA mutant. Results are presented as the log10 CFU g−1. Each data point represents a sample from an individual mouse, and horizontal bars indicate the medians. Two independent series of infections were performed: i (CFT073 WT and CFT073 ΔryfA) and ii (CFT073 WT and the complemented mutant). The ΔryfA mutant was attenuated 146-fold in bladder and 10,000-fold in kidneys compared to the WT parent strain. Data are means ± standard errors of the means of 10 mice. Statistical significance was calculated by one-way ANOVA (A and C) or Mann–Whitney Test (D): *, P < 0.05; **, P < 0.005; ***, P < 0.0001. NS, not significant.
Fig 6.
Role of RyfA during interaction with professional phagocytes.
Phagocytic cells were infected with CFT073 wild-type strain, ΔryfA or ΔoxyR isogenic mutants or the ΔryfA-complemented strain. (A) in vitro differentiated human monocyte-derived macrophages (HMDMs) and (B) THP-1 human macrophages were infected with different strains for 1 h, followed by gentamicin treatment. Cells were lysed and intracellular bacterial counts (CFU ml−1) were determined at different times post-infection (pi). Infection assessment of mCherry tagged CFT073 strains via flow cytometry in (C) HMDMs and (D) RAW264.7 cells. (E) Reactive oxygen species (ROS) production in RAW264.7 cells. RAW264.7 cells were infected at an MOI of 20 and intracellular generation of ROS was measured at 4 h and 6 h post-infection by using H2DCFDA. Cells were stained with 10 μM H2DCFDA. All assays were conducted in triplicate and repeated independently at least three times. The results are expressed as the means ± sem. Significant differences between mutant, WT and complemented mutant strains were determined using One-way ANOVA. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. NS, not significant.
Fig 7.
Role of RyfA during interaction with human macrophage cells.
Imaging flow cytometry analysis of human monocyte-derived macrophages (HMDMs) that were infected with CFT073 wild-type strain, ΔryfA mutant, the ΔryfA complemented strain and the ΔryfA fim L-ON strain at 2 h post-infection. (A) Representative images of HMDM singlets CD14+GFPhigh at 2h post-infection. (B) Infection assessment for double positive percentages of CD14+GFPhigh HMDMs. Below, representative flow cytometric histograms for an independent experiment. Results are expressed as the means ± sem of the replicate experiments. Significant differences between mutants, WT, and complemented mutant strain were determined using a paired Student t-test. *, P < 0.05.